Hydrogen exchange methods are being used to study allosteric regulatory mechanism in hemoglobin. Our thesis is that the essence of allosteric regulation is the way in which a protein like hemoglobin interconverts ligand binding energy and structure change energy. Therefore, to understand these mechanisms, it will be necessary to localize and measure structural free energy and free energy change throughout the protein. Concepts and methods based on hydrogen exchange (HX), developed under this grant, now appear able to accomplish this goal. The local unfolding model for protein H-exchange connects HX rate with structural free energy, and change in rate with change in energy. A functional labeling method uses H-T or H-D exchange to selectively label protein segments actively involved in any interaction being studied. The position of the label and the H-exchange behavior of the functionally sensitive NH can then be studied by use of a fragment separation method. With these approaches, three allostericauy important regions of hemoglobin have so far been identified. Some of their local bonding interactions have been demonstrated, their energy relationships have been studied, and remote, cross-subunit interactions have been detected. Hemoglobin's other allosterically involved regions will be sought and studied in these ways. Experiments will be done to measure the effects of allosteric effectors, defined chemical modifications, mutations, and partial heme liganding with O2 analogs. These effects will be measured at each of the allosterically involved positions, those local to the changes and those elsewhere in the protein, in order to map out the interaction network. New methods will be explored, including the application of mass spectrometry, chemical sequencing methods, and NMR to improve the fragment separation and functional labeling techniques. Other work directed at H-exchange mechanism and more generally at the physical nature of protein molecules will be continued, including a study of the ability of small molecules, especially analogs of water and hydroxide ion (the effective H-exchange catalyst), to penetrate into and migrate within proteins.